Targeting the COP9 signalosome for cancer therapy

The COP9 signalosome (CSN) is a highly conserved protein complex composed of 8 subunits (CSN1 to CSN8). The individual subunits of the CSN play essential roles in cell proliferation, tumorigenesis, cell cycle regulation, DNA damage repair, angiogenesis, and microenvironmental homeostasis. The CSN complex has an intrinsic metalloprotease that removes the ubiquitin-like activator NEDD8 from cullin-RING ligases (CRLs). Binding of neddylated CRLs to CSN is sensed by CSN4 and communicated to CSN5 with the assistance of CSN6, thus leading to the activation of deneddylase. Therefore, CSN is a crucial regulator at the intersection between neddylation and ubiquitination in cancer progression. Here, we summarize current understanding of the roles of individual CSN subunits in cancer progression. Furthermore, we explain how the CSN affects tumorigenesis through regulating transcription factors and the cell cycle. Finally, we discuss individual CSN subunits as potential therapeutic targets to provide new directions and strategies for cancer therapy.


Introduction
The ubiquitin-proteasome system (UPS) detects post-translational modification of proteins, and controls a wide range of cellular processes including protein degradation, signal transduction, transcriptional regulation, DNA repair, and cell cycle progression 1 . The degradation of proteins by the UPS begins with the addition of multiple ubiquitin molecules to lysine residues in proteins 2 . The binding of ubiquitin to substrate proteins is mediated by a cascade of enzymatic reactions involving E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases 3,4 . E3, acting at the end of the three-enzyme cascade, controls eukaryotic biological processes by promoting protein ubiquitination and degradation 5 . Cullin-RING ligases (CRLs), a superfamily of E3 complexes, are organized on the basis of various cullin scaffold proteins (CUL1, CUL2, CUL3, CUL4A, CUL4B, and CUL5) and use interchangeable substrate receptors to recruit various substrates onto a common catalytic scaffold 6 . CRLs are involved in the regulation of various dynamic cellular processes critical for cancer cell survival 7 . Cullins are modified by NEDD8, a well-studied ubiquitin-like protein that critical in a variety of cellular processes. Neddylation is the process of NEDD8 binding to a substrate protein, thereby promoting the E3 activity of CRLs and modulating protein activity and function 8,9 . Previous studies have demonstrated that remodeling of CRLs is initiated by the cleavage of NEDD8 from CRLs, catalyzed by the deneddylase COP9 signalosome (CSN) 10,11 .
The CSN, originally identified as a repressor complex of light activated development in Arabidopsis, is a multifunctional protein complex comprising 8 subunits (CSN1-CSN8) 12 . The biological function of the CSN is often determined through the regulation of CRLs, which are involved in the regulation of numerous cellular processes 13 . In the past few years, our laboratory has published articles exploring the mechanisms underlying the roles of the CSN in human cancers. In this review, we aim to summarize current understanding and discoveries made by our laboratory and others regarding all subunits of the CSN in cancer initiation and progression. We first briefly describe the structural and functional features of the CSN, a key regulator at the intersection between neddylation and ubiquitination that is associated with tumor progression. We then discuss critical issues including the roles of individual CSN subunits in cancer progression. We explain how the CSN affects tumorigenesis through regulating transcription factor stability and transcriptional activity. CSN-mediated regulation of cell cycle progression is also involved in tumorigenesis. In addition, we review the essential signaling pathways associated with CSN subunits and the potential therapeutic targets in human cancer. Finally, we discuss the CSN's emerging roles in carcinogenesis and highlight its promise as a potential target for cancer therapy.

Architecture and functional features of the CSN
COP9 is a multi-subunit protein complex that is a hallmark of eukaryotic cells and was first isolated from cauliflower 14,15 . The COP9 signalosome complex consists of 8 subunits (CSN1 to CSN8 from largest to smallest) 16 . Although CSN was initially identified as a repressor of photomorphogenesis in Arabidopsis 17,18 , the CSN subunit has been found to significantly affect several cellular functions, including angiogenesis, cell cycle control, DNA repair, maintenance of DNA fidelity, and microenvironmental homeostasis, all of which are essential in tumorigenesis 19,20 . Among the 8 CSN subunits, 6 CSN proteins (in the three-dimensional structure order of CSN7-CSN4-CSN2-CSN1-CSN3-CSN8) contain a proteasome lid-CSN-initiation factor 3 (PCI) domain characterized by helical repeats followed by a winged-helix subdomain 21 . The remaining subunits, CSN5, also known as Jun activation domain-binding protein 1 (Jab1/CSN5), and CSN6, are the only 2 Mpr1-Pad1-Nterminal (MPN) domain-containing subunits situated on top of the helical bundle formed by the C-terminal α-helices of each CSN subunit (Figure 1) 22 . Lingaraju et al. 23 have presented the crystal structure of the CSN and described the molecular architecture of the complex in detail. The crystal structure of the CSN reveals that the PCI helical repeat domains are responsible for binding SCFS KP2/CKS1 , whereas the helical bundle enables Jab1/CSN5 to sense the assembly state of the CSN. Jab1/CSN5 is a metalloprotease with a conserved Jab1 MPN domain metalloenzyme (JAMM) motif, and it requires a zinc ion as an activator. CSN6 is deficient in this JAMM motif and thus is identified as an MPN negative (MPN-) subunit, whereas Jab1/CSN5 is an MPN positive (MPN+) subunit 24,25 . As described in the introduction, the CSN regulates CRL activity by removing the covalently bound activator NEDD8. This deneddylation activity is performed by the JAMM/MPN+ domain of Jab1/CSN5, although this domain is inactive in isolation 24,26 . Binding of neddylated CRLs to CSN is sensed by CSN4 and communicated to CSN5 with the assistance of CSN6, thus resulting in the activation of deneddylase 23 . Furthermore, previous studies have indicated that the CSN complex is a critical platform enabling Jab1/CSN5 to function as a deubiquitinating enzyme (DUB). Moreover, the CSN controls the neddylation status of cells through destabilization of the associated deneddylation enzyme 1 (DEN1) and through its intrinsic DUB activity 27,28 . Therefore, the CSN is a key regulator at the intersection between neddylation and ubiquitination, which are associated with cancer progression. The following sections introduce all subunits of the CSN involved in tumor development to provide a foundation for cancer therapy.

The diverse roles of CSN subunits in cancer
A growing body of evidence indicates that CSN subunits play critical roles in various human cancers. We used the GEPIA database (http://gepia.cancer-pku.cn/index.html) to systematically assess the differential expression of CSN subunits in various types of cancers. In statistical analysis of the transcriptomic data of various cancers, the database identified the CSN gene expression profiles across various tumor samples and matched normal tissues. Here, we present only the types of human cancers with discrepancies in CSN expression (Supplementary Figure S1). Among the CSN subunits, Jab1/CSN5 is the most studied subunit that acts as an oncogene. However, evidence has indicated the involvement of CSN4 and CSN7 in human cancer. Herein, we describe recent advances in understanding of the roles of individual CSN subunits in cancer progression ( Table 1).

Dual roles of CSN1 in cancer
CSN1 plays a critical role in the ubiquitin-proteasome pathway and regulates various cellular processes, including the cell cycle and DNA repair 29 . Analysis of transcriptomic data from The Cancer Genome Atlas (TCGA) has revealed that CSN1 expression is enhanced in hepatocellular carcinoma (HCC) compared with normal tissue. High levels of CSN1 indicate poor prognosis in patients with HCC. Mechanistically, CSN1 promotes the migration and proliferation of HCC cells by upregulating cyclin A2 expression 30 . However, Feber et al. 31 have suggested that CSN1 is a novel tumor suppressor that disrupts miRNA-mediated gene silencing in penile squamous cell carcinoma when mutated. In addition, CSN1 has been reported to suppress mitogen-activated signal transduction 32 and has been implicated in the activation of p53 33 . However, few studies have attempted to explain the functional role of CSN1 in cancer. Therefore, the role of CSN1 in cancer development must be further defined.

Dual roles of CSN2 in cancer
CSN2 functions as a transcriptional corepressor and facilitates pluripotency maintenance 34 . CSN2 is considered a putative tumor suppressor gene and has diminished expression in tumor tissues 35 . Carvalho et al. 36 have shown that CSN2 might function as a tumor suppressor in colorectal cancer (CRC), and CSN2 could act as a candidate target gene of miR-15a-3p for preventing colorectal adenoma-to-carcinoma progression. Serum CSN2 levels serve as a prognostic marker in gastric cancer, and low serum CSN2 is associated with poor survival in patients with gastric cancer 37 . Furthermore, patients with low CSN2 expression have a higher risk of metastasis/recurrence. Multivariate Cox analysis has identified CSN2 as an independent prognostic factor for overall survival and disease-free  Regulates EMT-associated proteins 96 Gastric cancer Oncogene As a target of miR-146a, inhibits GPCR-mediated NF-κB activity 97 CSN, COP9 signalosome; HCC, hepatocellular carcinoma; EMT, epithelial-mesenchymal transition; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; TIMP-2, tissue inhibitor of metalloproteinase 2; RCC, renal cell carcinoma; GPCR, G protein-coupled receptor.

CSN3 as an oncogene in cancer
CSN3 is crucial for the maintenance of cell proliferation in mouse embryonic epiblasts and is associated with cancer progression and metastasis 41 . Knockdown of the CSN3 gene has been found to decrease the metastasis of osteosarcoma cells 42 . In addition, CSN3 knockdown suppresses metastasis of osteosarcoma cells to the lungs, both in vitro and in vivo 43 . The silencing of CSN3 also impairs EMT in osteosarcoma cells and prostate cancer 43, 44 . Furthermore, CSN3 has been reported to be a crucial regulator of cell survival and the cell cycle, and to mediate HCC cell proliferation 45 . CSN3 deletion restrains lung cancer tumor growth by repressing cell cycle progression 46,47 . Moreover, overexpression and amplification of CSN3 in osteosarcomas has been found to disrupt the anti-tumor pathway by targeting TP53 for proteasome-mediated degradation 48 . CSN3 may promote kidney cancer progression by regulating cyclin D1, caspase-3 expression and phospho-AKT (Thr308) 49 . Moreover, the CSN3 gene is located on chromosome 17p11.2~p12, an unstable chromosomal region that is frequently amplified in osteosarcomas and multiple myelomas [50][51][52][53][54] . Therefore, targeting CSN3 may be a promising strategy for anti-tumor therapy.

CSN4 overexpression in cancer
Few studies have investigated the role of CSN4 in cancer biology. Bhansali et al. 55 have demonstrated that CSN4 protein expression is robustly enhanced in prostate cancer. Furthermore, CSN4 promotes prostate cancer cell proliferation via down-regulating p53 protein and up-regulating sGCα1. In addition, TCGA database analysis has indicated differences in CSN4 mRNA levels between tumor and normal tissue samples. CSN4 alters apoptosis and proliferation in breast cancer cells and thereby promotes tumorigenesis 56 . Thus, the role and mechanism of CSN4 in human cancer require further exploration.

Jab1/CSN5 overexpression in cancer
Aberrant overexpression of Jab1/CSN5 has been implicated in many types of human malignancies, including gastric cancer 57,58 , breast cancer 59,60 , HCC 61 , non-small cell lung cancer 62 , osteosarcoma 63 , prostate cancer 64 , glioma 65 , laryngeal cancer 66 and many others 67 . Yuan et al. 68 have examined the differential expression of Jab1/CSN5 between various cancer tissues and corresponding normal tissue samples by querying the Oncomine database. Their results indicated overexpression of Jab1/CSN5 mRNA in central nervous system cancer, bladder cancers, myeloma, and breast cancer in 7 of 475 analyses. TCGA cohort and Gene Expression Omnibus dataset analyses have indicated that Jab1/CSN5 expression is significantly enhanced in cervical cancers compared with normal tissue. High Jab1/ CSN5 expression is associated with unfavorable clinical outcomes in patients with cervical cancer 69 . These statistical data indicate the role of Jab1/CSN5 as a biomarker and therapeutic target in numerous cancers. Furthermore, Jab1/CSN5 overexpression tends to correlate with cancer cellular proliferation 70 , vascular invasion 71 , lymph node metastasis 72 and histological differentiation clinical stage 73 . Indeed, Jab1/CSN5 promotes tumorigenesis via degrading several substrates, such as p53 74 , p27 75 , p14ARF 57 , Smad4 76 , and the WNT inhibitor DKK1 77 . These targets of Jab1/CSN5 function as tumor suppressors involved in various cellular processes, such as proliferation, apoptosis, angiogenesis, and the cell cycle 78 . Overall, marked progress has been made in verifying the important role of Jab1/CSN5 in tumor development. Jab1/CSN5 may serve as a novel biomarker of poor prognosis in human cancers.

CSN6 overexpression in cancer
CSN6 is overexpressed in many types of cancer, according to analysis of human cancer patient data sets from Gene Expression Omnibus and Oncomine 79  CSN6 gene copy number is positively correlated with tumor size 90 . Correspondingly, high CSN6 expression is significantly correlated with TNM stage, depth of invasion-pT status and lymph node metastasis-pN status in gastric cancer, breast cancer, and pancreatic adenocarcinoma 80,84,86 . Kaplan-Meier analysis has indicated that higher CSN6 expression is associated with shorter overall survival in patients with pancreatic cancer, HCC, or CRC 82,83,85 . In addition, CSN6 gene mRNA levels, assessed from TCGA data, have revealed higher CSN6 expression in tumor tissue than in normal tissue, and indicated that amplification of the CSN6 gene is associated with advanced disease stage 83 . Collectively, all these data suggest that CSN6 is a potential diagnostic biomarker and interference target for the treatment of human cancer.

CSN7A or CSN7B paralogs in cancer
CSN exists as 2 variant complexes containing either CSN7A or CSN7B paralogs that have overlapping functions in the deneddylation of CRL 91 . CSN7A mRNA and protein levels, evaluated by qRT-PCR and immunohistochemistry, are significantly lower in gastric cancer tissues than normal gastric tissues. Furthermore, lower CSN7A expression is associated with clinical manifestations, including positive lymph node metastasis and larger tumor size 92 . These data indicate the tumor-suppressive role of CSN7A in gastric cancer. According to data presented in Kaplan-Meier plotter and cBioPortal, CSN7B is associated with increased recurrence rates and decreased survival time in HCC and renal cell carcinoma (RCC) 93,94 . Furthermore, CSN7B deletion inhibits RCC cell invasion and proliferation 94 . Collectively, these studies suggest an oncogenic role of CSN7B in HCC and RCC.

CSN8 overexpression in cancer
Studies have revealed that CSN8 is a critical regulatory molecule that controls EMT, thus endowing CRC cells with vigorous invasion capability and highly aggressive metastatic characteristics 95 . Furthermore, CSN8 is elevated in cutaneous melanoma, and it accelerates cancer progression via regulation of EMT 96 . In addition, Crone et al. 97 have revealed that CSN8 is a target of microRNA-146a, which inhibits the activation of NF-κB-regulated tumor-promoting cytokines and growth factors in gastric cancer. These data indicate that CSN8 is an oncogene. However, the mechanisms underlying the role of CSN8 must be further explored.

Transcriptional regulation
The CSN was initially discovered as a transcriptional repressor of light-dependent growth in Arabidopsis 14 . This repression was attributed to the function of the CSN in regulating the stability of transcription factors that are usually unstable and degraded in darkness 98 . The CSN is structurally similar to the eukaryotic translation initiation factor eIF3 and is involved in the regulation of eIF3 99 . This section evaluates the role of the CSN as a transcriptional regulator in cancer. The CSN regulates transcription factor stability and transcriptional activity through the removal of the ubiquitin-like modifier NEDD8 from CRLs. For example, CSN promotes the deubiquitination of IκBα, thereby attenuating the activity of the transcription factor nuclear factor κB (NF-κB) 100,101 . Moreover, CSN2 and Jab1/CSN5 act as transcriptional corepressors and coactivators, respectively 102 . Jab1/CSN5 was initially identified as a transcriptional co-activator of c-Jun, which acts via activator protein 1 (AP-1) sites 103 . AP-1 transcription factors are associated with progression and recurrence of various cancers 104,105 . MK2-mediated phosphorylation of Jab1/CSN5 facilitates recruitment of Jun to AP-1 sites, thereby augmenting AP-1 activity in triple-negative breast cancer 106 . Recent studies have indicated the role of Jab1/CSN5 as a regulator of transcription factor activity. The transcription factor NF-κB is a critical regulator of many physiological and pathophysiological processes associated with carcinogenesis, including cell growth, apoptosis, and survival 107 . Jab1/CSN5 silencing may arrest cell cycle progression and inhibit invasion via decreasing the transcriptional activity of NF-κB in colorectal cancer 108 . Furthermore, Jab1/ CSN5 knockdown regulates signal transducer and activator of transcription 3 (STAT3) DNA-binding activity and increases STAT3 expression via protein-protein interaction in colon cancer cells 109 . E2F transcription factors are crucial regulators of cell cycle progression and cell fate control, and are induced by the E2F1 binding partner Jab1/CSN5. Increasing evidence indicates the role of Jab1/CSN5 in a variety of human cancers. Lu et al. 110 have suggested that the pro-proliferative function of Jab1/CSN5 may be attributable to E2F dependent apoptosis and mitosis. In addition, Jab1/CSN5 promotes the cytoplasmic localization and degradation of the transcription factor RUNX3, which plays crucial roles in fundamental cellular processes associated with tumor development 111 . Furthermore, Jab1/CSN5 promotes pancreatic cancer invasion and metastasis by stabilizing a Fos family protein [Forkhead box M1 (FOXM1)] 112 . The precise mechanism through which Jab1/ CSN5 regulates transcriptional activity remains to be investigated. Jun proteins have been suggested to bind Fos proteins to form the gene-regulatory protein AP-1 103 . Jab1/CSN5 or the holo CSN complex interacts with histone methyltransferases and chromatin, thereby regulating transcription 113,114 . The precise mechanism through which Jab1/CSN5 regulates transcriptional activity remains to be investigated.
CSN2, also known as Alien, was initially recognized as a corepressor of steroid hormone signaling 115 . CNS2 directly binds chromatin and enhances NAP-1-mediated nucleosome assembly, thereby contributing to gene silencing 116 . Moreover, CSN2 acts as a corepressor of E2F1 and is involved in cell proliferation 117 . In addition, CSN2 affects transcription factors through ubiquitination. For instance, NF-κB is required for the induction of CSN2, which in turn inhibits the ubiquitination and degradation of the transcription factor Snail 39 . Other subunits, such as CSN6, also attenuate transcription factor expression through ubiquitination in cancer cells 79,80,86 . However, the regulatory mechanisms of the CSN complex and its individual subunits in regulating gene expression remain to be further investigated.

Cell cycle
Multiple tumor types have been shown to be dependent on CSN-mediated modulation of cell cycle regulators (a finding that could potentially be used in anti-tumor therapies). The CSN and its subunits are involved in the regulation of cell-cycle progression (Figure 2). The first link between the CSN and cell cycle progression is that Jab1/CSN5 facilitates degradation of the cyclin-dependent-kinase inhibitor p27 and subsequent G1 phase arrest 118 . As an inhibitor of cyclin E-CDK2, p27 blocks the G1 to S transition in cell cycle progression and is a putative suppressor of tumorigenesis [119][120][121] . In addition, the cytoplasmic shuttling of p27 is associated with poor survival in patients with cancer 122,123 . The CSN complex and CSN5/CSN6 induce the cytoplasmic translocalization and subsequent degradation of p27 124,125 . Indeed, accumulating evidence indicates that Jab1/CSN5 upregulation is inversely correlated with p27 levels and is associated with poor survival in several human cancers 75,108 . The CSN also plays an essential role in the regulation of other cell cycle-associated gene expression. For instance, Jab1/CSN5 promotes the ubiquitination and degradation of p53 and antagonizes the transcriptional activity of p53 126,127 . CSN6 facilitates cancer cell growth through MDM2-mediated p53 degradation 128,129 . CSN acts upstream of the ubiquitin E3 ligase Skp2, which controls G1⁄S cell cycle regulators 130 . CSN6 promotes Skp2-mediated degradation of the cyclin-dependent kinase inhibitor p57 131 , whereas Jab1/CSN5 promotes p57 degradation independently of Skp2 in HCC 61 . In addition to the ubiquitin-dependent degradation of proteins, Jab1/CSN5 and CSN6 promote tumorigenesis via ubiquitin independent proteasomal degradation of the cell cycle regulators p16 INK4a and p14ARF 57,84 .
Beyond Jab1/CSN5 and CSN6, other CSN subunits are involved in the regulation of cell cycle progression. CSN2 disruption causes cell proliferation deficiency through the accumulation of p53, cyclin E, and the cyclin-dependent kinase inhibitor p21 Cip1/Waf1132 . CSN2 also restrains p27 kip1 degradation and blocks G1/S phase progression through deneddylation of SCF Cul1 133 . In lung cancer, CSN3 knockdown blocks cell cycle progression at G0/G1 phase by upregulating p21 and downregulating CDK4 and cyclin B1 46 . In summary, the CSN controls multiple cell cycle regulators and steps in cell cycle progression. Therefore, elucidating the CSN subunits and the subcomplexes' function in cell cycle regulation will be essential.

Associated signaling pathways of CSN subunits
The CSN lies at the intersection of a range of signaling pathways believed to be critical to tumor development. The network of protein interactions involving CSN is highly complex. The interaction database IntAct (http://www.ebi.ac.uk/intact/) currently lists 7,656 binary interactions for the COP9. To graphically illustrate at least part of this interaction network, we performed a STRING database search (http://stringdb. org/) for proteins interacting either functionally or physically with 8 individual CSN subunits as the input (Figure 3). Recent studies have demonstrated that the CSN is involved in several signaling pathways critical to tumor development. Herein, we describe the roles of the CSN in these signaling pathways (Figure 4) to improve understanding of tumorigenesis.

Wnt/β-catenin signaling
The Wnt/β-catenin signaling pathway has crucial roles in angiogenesis and cell proliferation. The CSN regulates Wnt/βcatenin signaling by targeting β-catenin for degradation by the UPS, thus controlling the balance between β-catenin and APC in HeLa cells. Disruption of this balance leads to cancer by promoting cell transformation and metastasis 134 . Wnt/βcatenin signaling is critical to the progression and development of CRC. Jab1/CSN5 depletion affects Wnt signaling by downregulating β-catenin and increases the secretion of the Wnt inhibitor DKK1 in CRC cells 77,135 . In addition, Jab1/ CSN5 positively regulates the expression of β-catenin via the E3 ubiquitin ligase SIAH-1 136,137 . Thus, these data suggest that Jab1/CSN5 is a promising target for CRC therapy. CSN6 also stabilizes β-catenin expression and facilitates EMT in papillary thyroid cancer cells. Furthermore, CSN6 knockdown sensitizes papillary thyroid cancer cells to FH535 therapy through downregulation of the Wnt/β-catenin signaling pathway 138 .

PI3K/AKT signaling
PI3K/AKT signaling pathways are frequently perturbed in human cancers and are involved in tumor processes including cell differentiation, growth, and development 139 . A recent study has demonstrated that Jab1/CSN5 enhances epidermal growth factor receptor (EGFR) stability by decreasing EGFR ubiquitination, thereby activating the PI3K/AKT signaling pathway in osteosarcoma cells 140 . Additionally, Jab1/CSN5 silencing may arrest cell cycle progression and inhibit invasion via the PI3K/AKT/NF-κB signaling pathway in CRC 108 . Furthermore, macrophage migration inhibitory factor (MIF) is an important inflammatory cytokine involved in tumorigenesis. Lue et al. 141 have found that Jab1/CSN5 prevents MIF secretion, thereby modulating autocrine MIF-mediated PI3K/ AKT signaling in cancer cells. Moreover, PI3K/AKT signaling stimulates the synergistic interaction between Jab1/CSN5 and LASP1, thus promoting CRC progression 72 .

HER-2 and EGFR signaling
HER-2 plays a critical role in the transformation and growth of cancers in many malignancies, and is associated with poor prognosis in human cancers 142,143 . HER-2 enhances Jab1/ CSN5 expression through transcriptional activation in breast cancer 144 . HER-2 transcriptionally increases Jab1/CSN5 promoter activity through the AKT/β-catenin pathway, thus promoting the proliferation of breast cancer cells 145 . In addition, the HER2-AKT signaling pathway positively regulates the stability of CSN6 protein via regulating ubiquitin-proteasomal degradation during carcinogenesis 129 . Furthermore, HER-2 encodes a transmembrane tyrosine kinase receptor with extensive homology to EGFR 146 . EGFR-ERK signaling upregulates CSN6 and subsequently enhances programmed cell death ligand-1 (PD-L1) stability in glioblastoma 147 . Moreover, CSN6 positively regulates EGFR stability, thereby promoting glioblastoma proliferation and metastasis 88 . Jab1/CSN5 is also a target of EGFR signaling, and activation of EGFR promotes the translocation of Jab1/CSN5 from the cytoplasm to the nucleus in breast cancer cells 148 . Together, CSN6-associated HER-2 and EGFR signaling may be potential therapeutic targets in cancer.

ERK and MAPK signaling
The ERK/MAPK signaling pathway is one of the most important pathways involved in cell proliferation. MAPK signaling is downstream of various growth factor receptors, including EGFR 149

MDM2/p53 signaling
MDM2, a primary cellular inhibitor of p53, is frequently overexpressed in human cancers 150 . CSN-specific phosphorylation targets p53 for ubiquitin-26S proteasome-dependent degradation mediated by MDM2 33 . CSN6 enhances MDM2 stabilization and plays an essential role in promoting tumorigenesis by regulating MDM2/p53 signaling 90 . On the basis of previous studies, we propose that CSN6-mediated stabilization of MDM2 leads to ubiquitination-dependent degradation of p53, thereby interfering with the tumor-suppressive role of p53. Correspondingly, Jab1/CSN5 facilitates MDM2-mediated p53 degradation and promotes p53 nuclear export. Moreover, Jab1/CSN5 overexpression leads to the stabilization of MDM2 and antagonizes the transcriptional activity of p53 in human cancer 127 . Therefore, the CSN is important for modulating p53-mediated tumor suppression by controlling the MDM2/ p53 signaling pathway.

STAT3 signaling
Signal transducer and activator of transcription (STAT) proteins play critical roles in regulating fundamental cellular processes including cell growth and differentiation. Among the STATs, abnormal activation of STAT3 is associated with a variety of human malignancies 151 . Jab1/CSN5 regulates unphosphorylated STAT3 DNA-binding activity via protein-protein interaction in colon cancer cells. Likewise, Jab1/CSN5 silencing decreases the expression of STAT3 target genes 109 . Consistently, STAT3 silencing decreases the expression of Jab1/CSN5 and inhibits Jab1/CSN5 promoter activity in cancer cell lines 152 . Furthermore, treatment with the cytokine IL-6 enhances Jab1/CSN5 expression, but this effect is blocked by inhibition of STAT3. These findings indicate that the IL-6/ STAT3 signaling pathway is involved in the activation of Jab1/ CSN5 transcription 153 . Therefore, the relationship between Jab1/CSN5 and STAT3 associated with carcinogenesis may enhance understanding of the regulatory mechanism of Jab1/ CSN5 in human cancer.

NF-κB signaling
The NF-κB family of transcription factors are key regulators of cell survival and tumor proliferative signaling pathways 154 . NF-κB induced expression of Jab1/CSN5 leads to PD-L1 stabilization and immune suppression in cancer cells 155 . Additionally, NF-κB induced expression of CSN2 blocks the ubiquitination and degradation of the Snail transcription factor, which is required for cell migration and invasion mediated by inflammation 39 . Moreover, activation of NF-κB is necessary for the upregulation of CSN2, which in turn regulates Snail stabilization via blocking its interaction with β-TrCP and GSK-3β 156 . In CRC, Jab1/CSN5 knockdown inhibits the secretion of NF-κB and restrains cell proliferation via the PI3K/AKT/NF-κB signaling pathway 108 .
In gastric cancer, COPS7A promotes IκBα deubiquitination via CSN-associated deubiquitinase USP15, then inactivates NF-κB signaling 92 . Thus, CSN-mediated NF-κB signaling may serve as a potential target for the treatment of human cancers in the future.

HIF-1α signaling
Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes involved in angiogenesis, cell invasion, and survival. HIF-1α overexpression is associated with elevated patient mortality in many cancer types 157 . CSN increases HIF-1α degradation by promoting the dissociation of HIF-1α from its oxygen-dependent regulator Von Hippel-Lindau (pVHL) in cells 158 . However, Jab1/CSN5 interacts with both HIF-1α and pVHL, thereby stabilizing HIF-1α and positively regulating HIF function 159 . Furthermore, CSN8 partially regulates hypoxia-induced EMT and dormancy by activating the HIF-1α signaling pathway, and it enhances HIF-1α mRNA expression via activating NF-κB, thus endowing CRC cells with metastatic and invasive abilities 95 .

CSN as a therapeutic target
CSN overexpression has been suggested to be generally associated with tumor development in humans, and the development of specific CSN inhibitors is expected to have remarkable effects on cancer treatment. Targeting the CSN may be a productive way to hinder metastasis and proliferation of malignant tumors, and improve chemotherapy and radiotherapy. The CSN extracted from human erythrocytes has kinase activity and phosphorylates proteins as a consequence of degradation through the ubiquitin pathway. The CSN-associated kinase activity is inhibited by curcumin, emodin, resveratrol, and DRB 160 . Curcumin and emodin also inhibit CSNassociated kinases that trigger proteasome-dependent degradation of Id1 and Id3 161 . Id1 and Id3 are regulators of tumor angiogenesis that are stimulated by CSN-directed c-Jun signaling 162,163 . Among the inhibitors, curcumin is the most studied drug associated with the inhibition of CSN. Curcumin is a yellow plant pigment that induces tumor cell death and apoptosis via the inactivation of CSN 164 . Li et al. 165 have generated a water-soluble polyethylene glycol-conjugated curcumin that inhibits pancreatic cancer cell proliferation by activating Jab1/ CSN5. Moreover, this compound sensitizes pancreatic cancer cells to gemcitabine and could potentially be developed as an anti-tumor agent. T83, a novel curcumin analog that induces G2/M arrest and apoptosis, exhibits anticancer activity and induces radiosensitivity through inactivation of Jab1 in nasopharyngeal carcinoma 166 . Another potential target drug is troglitazone, which directly inhibits Jab1/CSN5 promoter activity by repressing Tcf4-and Sp1-mediated transcription. Ectopic expression of Jab1/CSN5 counteracts troglitazone-induced growth inhibition. Animal studies have verified that troglitazone decreases Jab1/CSN5 expression and suppresses HCC cell growth in tumor tissues 167 . These results provide insights into how CSN may be a potential target for anti-tumor therapy. However, drugs targeting the holo CSN complex and other CSN subunits must be further investigated.
Various types of cancers express high levels of PD-L1 and exploit programmed cell death-1 (PD-1)/PD-L1 signaling to evade T cell immunity. Immune checkpoint-blockade treatments targeting PD-1/PD-L1 have consistently shown remarkable anti-tumor effects in patients with advanced cancers 154,168 . Hence, better understanding of the regulatory mechanisms of PD-L1 should provide substantial benefits to patients in cancer diagnosis and immunotherapy. CSN6 and Jab1/CSN5 inhibit the degradation of PD-L1 and subsequently sustain PD-L1 stability in cancer cells 147,155 . In addition, inhibition of Jab1/CSN5 by curcumin decreases the expression of PD-L1 and sensitizes cancer cells to anti-CTLA4 therapy 155 . The anti-inflammatory drug berberine is a negative regulator of PD-L1 and enhanced the sensitivity of tumor cells to co-cultured T-cells. Berberine decreases the expression of PD-L1 and promotes antitumor immunity through inhibiting Jab1/CSN5 activity 169 . Therefore, the relationship between CSN and PD-L1 may enhance understanding of their regulatory mechanism through immune evasion and lead to the development of an efficient cancer therapeutic drug. CSN is involved in chemotherapy and radiotherapy resistance. CSN2 knockdown suppresses the expression of Snail and enhances the sorafenib sensitivity of HCC cells 170 . Likewise, Jab1/CSN5 silencing reverses the sorafenib resistance of HCC cells and downregulates multi-drug-resistance proteins, including adenosine triphosphate binding cassette (ABC)B1, ABCG2, and ABCC2. Furthermore, repression of Jab1/CSN5 sensitizes cancer cells to cisplatin and ionizing radiation 171 . Together, these findings support that targeting CSN may emerge as a novel therapeutic approach for cancer treatment. Thus, developing effective CSN specific inhibitors for clinical cancer therapy should prove meaningful. In addition, combined treatment with small molecular inhibitors of the associated signaling pathways involving CSN may be a good strategy for the treatment of human cancers.

Conclusions and perspectives
The CSN, a critical regulator at the intersection between neddylation and ubiquitination, is associated with tumor development. The CSN controls neddylation status of cells by destabilizing the associated DEN1 and through its intrinsic DUB activity. In this review, we discussed the roles of the CSN in cancer, which may be associated with proteasome-mediated protein degradation activity. Recent data (Figure 5) have implicated the CSN in a range of cellular processes relevant to cancer progression, such as transcriptional regulation, cell cycle control, and immune evasion. The CSN has multiple prominent functions that affect multiple targets and signaling pathways, which are often carcinogenic. The CSN is activated by a diverse complex network of signaling pathways, which influence one another. Although the CSN subunits have dual roles in tumor development, inhibition of the CSN may be a valuable strategy for the treatment of certain types of cancer.
Current CSN studies based on molecular biology and model organisms support the diverse roles of this complex in cancer. However, a paradox is evident in multiple published studies indicating that the CSN is critical for tumor development. For example, CSN1 and CSN2 have dual roles in cancer progression as oncogenes or tumor suppressors. CSN7B deletion inhibits cell invasion and proliferation, whereas CSN7A is a tumor suppressor. Furthermore, CSN promotes cyclin-dependent kinase inhibition in a manner dependent on or independent of Skp2-mediated degradation in cancer cells. Thus, the detailed mechanistic regulation of the CSN in cancer remains to be investigated. Additional studies are needed to further dissect the function of the CSN and the individual subunits in cancer. Identifying the upstream signals and downstream substrates of the CSN is necessary before efficient therapeutic strategies targeting the CSN can be developed. Crucially, a drug screening approach might be required to develop new inhibitors of the CSN for cancer therapy.